Biology Reference
In-Depth Information
2.1.2.2
Protocol
The RAR
a
LBD and the RXR
a
LBD cloned, respectively, into the pET-15b vector
(as a histidine-tagged protein) or into the pET-3a vector are expressed in
E. coli
cells.
For both transformants, cells are grown on LB plates containing 50
m
g ampicillin/
mL. A 3 mL LB starter culture containing 200
m
g ampicillin/mL is inoculated with
RAR LBD/pET-15b- or RXR LBD/pET-3a-transformed BL21(DE3) grown over-
night at 37
C, and 12 mL is used to inoculate 500 mL of LB containing 200
m
g
ampicillin/mL. Cultures are grown at 37
C to an OD600 of 0.4-0.5, and expression
of T7 RNA polymerase is induced by the addition of IPTG to 1 mM. After an addi-
tional incubation for 2.5 h at 25
C, cells are harvested by centrifugation. The pellets
from 3 L of RAR
a
LBD and 2 L of RXR
a
LBD cultures are suspended together in
62.5 mL of ice-cold buffer A (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl,
pH 8.0). The suspension is sonicated for 2 min, diluted with 62.5 mL of ice-cold
buffer A, and sonicated again. The crude extract is obtained by centrifugation for
30 min at 40,000 rpm. A 5 mL HisTrap chelating column is equilibrated with 10 vol-
umes of sterile deionized water, 50 mM NiSO4, sterile deionized water, and finally
10 volumes of buffer A. The crude extract is passed over the column at a flow rate
of 5 mL/min, followed by washing with 20 volumes of buffer A and 20 volumes of
50 mM imidazole in buffer A. The heterodimer is eluted with 20 volumes of buffer B
(150 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). Fractions are then
analyzed by SDS-polyacrylamide gel electrophoresis, pooled, dialyzed against
5 mM dithiothreitol, 150 mMNaCl, and 10 mMTris-HCl, pH 7.5, and concentrated.
The heterodimer is further purified by gel filtration using a HiLoad 26/60 Superdex
75 column preequilibrated with the same buffer. Fractions of 2 mL are analyzed
by SDS-polyacrylamide gel electrophoresis, pooled, and concentrated to 5 mg/
mL (Centriprep 30) for subsequent
in vitro
analysis. At this step, the structural ho-
mogeneity of the preparations is evaluated by electrophoretic analysis using
nondenaturating conditions (native gels), dynamic light scattering (DLS), or circular
dichroism (CD).
2.1.3
Monitoring NR-NR interactions by noncovalent electrospray
ionization mass spectrometry
Since the 1990s, electrospray ionization mass spectrometry (ESI-MS) has been reg-
ularly used to study noncovalent complexes and offers new possibilities for the study
of such complexes, providing direct evidence for their formation and an accurate de-
termination of their binding stoichiometry. For instance, ESI-MS was previously
used to detect the ER
a
LBD in its dimeric noncovalently bound form (
Witkowska
et al., 1997; Witkowska, Green, Carlquist, & Shackleton, 1996
) or RXR
a
-RAR
a
heterodimers (
Sanglier et al., 2004
). Hence, supramolecular mass spectrometry is
a powerful tool to rapidly and unambiguously determine if two NRs can interact.
The following protocol is an example given for the use of ESI-MS under nondena-
turating conditions to monitor the interaction between the RXR
a
and RAR
a
LBDs
(
Fig. 2.4
).